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Background: Von Willebrand factor (VWF) is a multimeric glycoprotein that plays important roles in hemostasis and thrombosis. C-terminal interchain-disulfide bonds in the cystine knot (CK) domain are essential for VWF dimerization. Previous studies have reported that missense variants of cysteine in the CK domain disrupt the intrachain-disulfide bond and cause type 3 von Willebrand disease (VWD). However, type 3 VWD-associated noncysteine substitution variants in the CK domain have not been reported. Objective: To investigate the molecular mechanism of a novel non-cysteine variant in the CK domain, VWF c.8254 G>A (p.Gly2752Ser), which was identified in a patient with type 3 VWD as homozygous. Methods: Genetic analysis was performed by whole exome sequencing and Sanger sequencing. VWF multimer analysis was performed using SDS-agarose electrophoresis. VWF production and subcellular localization were analyzed using ex vivo endothelial colony forming cells (ECFCs) and an in vitro recombinant VWF (rVWF) expression system. Results: The patient was homozygous for VWF-Gly2752Ser. Plasma VWF enzyme-linked immunosorbent assay showed that the VWF antigen level of the patient was 1.2% compared with healthy subjects. A tiny amount of VWF was identified in the patient's ECFC. Multimer analysis revealed that the circulating VWF-Gly2752Ser presented only low molecular weight multimers. Subcellular localization analysis of VWF-Gly2752Ser-transfected cell lines showed that rVWF-Gly2752Ser was severely impaired in its ER-to-Golgi trafficking. Conclusion: VWF-Gly2752Ser causes severe secretory impairment because of its dimerization failure. This is the first report of a VWF variant with a noncysteine substitution in the CK domain that causes type 3 VWD.

Hemophilia B (HB) is a hereditary bleeding disorder caused by the genetic variation of the coagulation factor IX (FIX) gene (F9). Several F9 structural abnormalities, including large deletion and/or insertion, have been observed to cause HB development. However, there is limited information available on F9 deep intronic variations. In this study, we report about a novel large deletion/insertion observed in a deep region of F9 intron 1 that causes mRNA splicing abnormalities.The patient was a Japanese male diagnosed with moderate HB (FIX:C = 3.0 IU/dL). The genomic DNA of the patient was isolated from peripheral blood leukocytes. DNA sequences of F9 exons and splice donor/acceptor sites were analyzed via polymerase chain reaction and Sanger sequencing. Variant-affected F9 mRNA aberration and FIX protein production, secretion, and coagulant activity were analyzed by cell-based exon trap and splicing-competent FIX expression vector systems.A 28-bp deletion/476-bp insertion was identified in the F9 intron 1 of a patient with moderate HB. A DNA sequence identical to a part of the inverted HNRNPA1 exon 12 was inserted. Cell-based transcript analysis revealed that this large intronic deletion/insertion disrupted F9 mRNA splicing pattern, resulting in reduction of protein-coding F9 mRNA.A novel deep intronic F9 rearrangement was identified in a Japanese patient with moderate HB. Abnormal F9 mRNA splicing pattern due to this deep intronic structural variation resulted in a reduction of protein-coding F9 mRNA, which probably caused the moderate HB phenotype.

CEBPA-IGH, a fusion gene of the immunoglobulin heavy-chain locus (IGH) and the CCAAT enhancer-binding protein α (C/EBPα) gene, is recurrently found in B-ALL cases and causes aberrant expression of C/EBPα, a master regulator of granulocyte differentiation, in B cells. Forced expression of C/EBPα in B cells was reported to cause loss of B-cell identity due to the inhibition of Pax5, a master regulator of B-cell differentiation; however, it is not known whether the same mechanism is applicable for B-ALL development by CEBPA-IGH. It is known that a full-length isoform of C/EBPα, p42, promotes myeloid differentiation, whereas its N-terminal truncated isoform, p30, inhibits myeloid differentiation through the inhibition of p42; however, the differential role between p42 and p30 in ALL development has not been clarified. In the present study, we examined the effect of the expression of p42 and p30 in B cells by performing RNA-seq of mRNA from LCL stably transfected with p42 or p30. Unexpectedly, suppression of PAX5 target genes was barely observed. Instead, both isoforms suppressed the target genes of MEF2 family members (MEF2s), other regulators of B-cell differentiation. Similarly, MEF2s target genes rather than PAX5 target genes were suppressed in CEBP-IGH-positive ALL (n = 8) compared with other B-ALL (n = 315). Furthermore, binding of both isoforms to MEF2s target genes and the reduction of surrounding histone acetylation were observed in ChIP-qPCR. Our data suggest that the inhibition of MEF2s by C/EBPα plays a role in the development of CEBPA-IGH-positive ALL and that both isoforms work co-operatively to achieve it.

Hereditary thrombophilia is a condition in which individuals are susceptible to the formation of thrombi due to a hereditary deficiency in anticoagulant factors, antithrombin (AT), protein C (PC), or protein S (PS). Many Japanese thrombophilia patients have PS deficiency, especially PS p.K196E (also called as PS Tokushima), which is exclusive to the Japanese population, and thrombosis sometimes occurs during pregnancy. At present, no management guidelines for pregnancy and delivery in thrombophilia patients have been developed. The Study Group for Hereditary Thrombophilia, one of the research groups of blood coagulation abnormalities in the Research Program on Rare and Intractable Diseases supported with the Research Grants of the Ministry of Health, Labour and Welfare Science, has therefore developed this clinical guidance to provide healthcare workers with necessary information on safe pregnancy, parturition and neonatal management, adopting a format of responses to seven clinical questions (CQ). At the end of each answer, the corresponding Recommendation Level (A, B, C) is indicated.

Background Coagulation factor XI (FXI) is a plasma serine protease zymogen that contributes to hemostasis. However, the mechanism of its secretion remains unclear. Objective To determine the molecular mechanism of FXI secretion by characterizing a novel FXI mutant identified in a FXI-deficient Japanese patient. Patient/methods The FXI gene (F11) was analyzed by direct sequencing. Mutant recombinant FXI (rFXI) was overexpressed in HEK293 or COS-7 cells. Western blotting and enzyme-linked immunosorbent assay were performed to examine the FXI extracellular secretion profile. Immunofluorescence microscopy was used to investigate the subcellular localization of the rFXI mutant. Results We identified a novel homozygous frameshift mutation in F11 [c.1788dupC (p.E597Rfs*65)], resulting in a unique and extended carboxyl-terminal (C-terminal) structure in FXI. Although rFXI-E597Rfs*65 was intracellularly synthesized, its extracellular secretion was markedly reduced. Subcellular localization analysis revealed that rFXI-E597Rfs*65 was abnormally retained in the endoplasmic reticulum (ER). We generated a series of C-terminal-truncated rFXI mutants to further investigate the role of the C-terminal region in FXI secretion. Serial rFXI experiments revealed that a threonine at position 622, the fourth residue from the C-terminus, was essential for secretion. Notably, Thr622 engages in the formation of an α-helix motif, indicating the importance of the C-terminal α-helix in FXI intracellular behavior and secretion. Conclusion FXI E597Rfs*65 results in the pathogenesis of a severe secretory defect due to aberrant ER-to-Golgi trafficking caused by the lack of a C-terminal α-helix motif. This study demonstrates the impact of the C-terminal structure, especially the α-helix motif, on FXI secretion.

Most clotting factors were initially discovered as agents functionally deficient in the plasmas of rare patients with hereditary coagulation disorders. During 1940s to 1960s, many factors were named by different investigators after the name of the patient who lacked a new factor. Consequently, there were the same factors with different names. To avoid confusion, the International Committee on the nomenclature of clotting factors was founded and discussed the identity or non-identity of clotting factors by specialists. There remain, however, several factors that were not officially authorized. We attempt to review some of these factors that seem to be not identical with any known clotting factors.

Inhibitor-development is a serious complication in patients with haemophilia (PwH). Previous studies reported that therapeutic and genetic factors could be associated with these alloantibodies. Relevant clinical features such as genetic-background and different treatment regimens in Japan remain unclear, however.To analyse a nation-wide Japanese registry for PwH, and to examine risk factors for inhibitor-development.Newly diagnosed patients with haemophilia A (PwHA) or haemophilia B (PwHB) without inhibitors after 2007, and with treatment records traceable from 0 to 75 exposure days (ED), were enrolled in the Japan Hemophilia Inhibitor Study 2 (J-HIS2) initiated in 2008. Of 417 patients (340 PwHA, 77 PwHB) from 46 facilities, 83 (76 PwHA, 7 PwHB) were recorded with inhibitors by July 2020. Inhibitors were observed in 31.0% of severe PwHA, 8.0% moderate and 1.6% mild and in 17.1% of severe PwHB. The majority of inhibitors (89.7% in severe PwHA and 71.4% in severe PwHB) were detected on or before 25ED (median 12ED in PwHA and 19ED in PwHB). Genotyping in these severe patients identified an association between inhibitor-development and null variants of F8 (P .01) or F9 (P .05). A lower incidence of inhibitors was recorded in severe PwHA treated with prophylaxis than in those treated on-demand (P .01). A past-history of intracranial-haemorrhage appeared to be associated with inhibitor-development, while FVIII-concentrates infusion and routine vaccination on the same day was not related to inhibitor-development.The J-HIS2 study has identified significant clinical variables associated with inhibitor-development in Japanese PwH, consistent with other global studies.

Introduction Factor VIII activity (FVIII:C) is measured by one-stage clotting assay (OSA) or chromogenic substrate assay (CSA). Significant differences in FVIII:C between OSA (FVIII:C1st ) and CSA (FVIII:CChr ) are described as assay discrepancy in nonsevere haemophilia A (HA). A large number of reagent combinations (APTT reagent and FVIII-deficient plasma) are used for OSA, but the impact of variations in reagent combinations on assay discrepancy has not been fully characterized. Aim To clarify the variations in FVIII:C1st /FVIII:CChr ratios according to OSA reagent combination in HA subjects with/without assay discrepancy. Methods Thirty-nine patients previously diagnosed with nonsevere HA were enrolled, and their FVIII genes were investigated and FVIII:C levels were assessed by a single CSA reagent and 11 OSA reagent combinations. Receiver operating characteristic (ROC) curve analysis was used to predict possible cut-off values of the FVIII:C1st /FVIII:CChr ratio to define FVIII assay discrepancy for each reagent combination. Results Patients were categorized into nondiscrepant (n = 25), discrepant (n = 5) and unclassified (n = 9) groups according to their genotypes and information in the database. The FVIII:C1st /FVIII:CChr ratio in nondiscrepant HA varied widely, depending on the APTT reagents and FVIII-deficient plasma used. The ratio in discrepant HA patients differed with respect to their genotype and the reagent combination used. ROC curve analyses revealed that cut-off values to distinguish the assay discrepancy differed depending on the reagents used, but revealed two novel genotype variants, p.Cys573Gly and p.Gly582Arg, associated with FVIII assay discrepancy. Conclusion Our findings showed that the FVIII:C1st /FVIII:CChr ratio is dependent on the reagent combination used for OSA.

Protein S is a vitamin K-dependent glycoprotein with important anticoagulant, fibrinolytic, anti-inflammatory, anti-apoptotic, and cytoprotective functions. Congenital protein S deficiency is an autosomal dominant thrombophilia due to protein S gene (PROS1) variations. Our group identified a variation in PROS1 that translates into protein S deficiency: c.50 T C (p.Leu17Pro). Here, we investigated the mechanisms by which this variation results in protein S deficiency.The effect of L17P substitution on protein S signal peptide was predicted by in silico (a computational prediction technique) analysis of hydrophobicity and signal peptide cleavage. Recombinant protein S was overexpressed in HEK293 and COS-7 cells. Intracellular kinetics and extracellular secretion of recombinant protein S-L17P were analyzed by western blotting and immunocytochemistry.In silico hydrophobicity analysis showed that protein S-L17P had disrupted hydrophobic status in the h-region of its signal peptide. Under normal culture conditions, recombinant protein S -L17P was not detected in either transfectant cell lysates or medium. Upon treatment with a proteasome inhibitor, recombinant protein S-L17P was clearly detected in the cell lysate, but not in the culture medium. Recombinant protein S-L17P did not undergo post-translational modification with N-glycosylation, suggesting that the nascent polypeptide of recombinant protein S-L17P is not transported to the endoplasmic reticulum lumen, but is mislocalized to the cytosol.PROS1-L17P variation translates into protein S deficiency. Protein S-L17P causes its cytosolic mislocalization resulting in its proteasome-dependent degradation.

Bone marrow development and endochondral bone formation occur simultaneously. During endochondral ossification, periosteal vasculatures and stromal progenitors invade the primary avascular cartilaginous anlage; this induces primitive marrow development. We previously determined that bone marrow podoplanin (PDPN)-expressing stromal cells exist in a perivascular microenvironment, and promote megakaryopoiesis and erythropoiesis. In this study, we aimed to examine the involvement of PDPN-expressing stromal cells in the postnatal bone marrow generation. We found that periosteum-derived PDPN-expressing stromal cells regulate vascularization during postnatal epiphyseal marrow development. Our findings suggest that these cells act as pericytes on the primitive vasculature of the nascent marrow. They invade the cartilaginous epiphysis and regulate marrow development and homeostasis by maintaining vascular integrity. To the best of our knowledge, this is the first study to comprehensively examine how PDPN-expressing stromal cells contribute to marrow development and homeostasis.

ファイル公開：2021-04-01

We managed perioperative hemostasis for a 72-year-old man with hemophilia A and low inhibitor titers (3 BU/mL), who underwent osteosynthesis for supracondylar fracture of the left humerus. He was treated perioperatively using the combination of high doses of factor VIII (FVIII) with recombinant human Factor VIII Fc fusion protein (rFVIIIFc), followed by emicizumab. On the day of surgery (day 0), he was administered bolus infusion of 150 IU/kg rFVIIIFc, followed by continuous infusion at a dose of 4 IU/kg/h. Emicizumab, 3 mg/kg, was injected subcutaneously once a week, on days 5, 12, 19, and 26. Inhibitors were detected on day 6 at a titer of 4 BU/mL and FVIII:C decreased to below assay sensitivity limits on day 10. The rate of increase in inhibitor titers was high, with inhibitors increasing to 343.4 BU/mL on day 14. The transition of thrombin production by thrombin generation assay (TGA) showed temporary decrease in thrombin production on day 7, although it was restored by day 10, i.e., five days after commencement of emicizumab therapy. Rotational thromboelastometry displayed consistent results with TGA, showing that clotting time was prolonged and the alpha angle decreased to less than measurable levels on day 6, although they were improved by day 10. There were no bleeding-related events or other adverse events throughout the perioperative period. In conclusion, emicizumab was effective for the management of perioperative hemostasis after development of an anamnestic response in a patient with hemophilia A with inhibitors. Combination therapy with high doses of FVIII followed by emicizumab could be a workable alternative for patients with hemophilia A with inhibitors.

Background Congenital antithrombin (AT) deficiency, which arises from various SERPINC1 defects, is an autosomal-dominant thrombophilic disorder associated with a high risk of recurrent venous thromboembolism. Patients/methods We investigated SERPINC1 defects in Japanese patients with congenital AT deficiency who developed venous thromboembolism or had a family history of deep vein thrombosis. We analyzed the full DNA sequences of SERPINC1 exons and exon-intron junctions by PCR-mediated direct sequencing. If no mutation was found, multiplex ligation-dependent probe amplification (MLPA) was conducted for the relative quantification of the copy number of all exons in SERPINC1. If splice-site mutations were detected, mRNA splicing abnormalities were further investigated using an in vitro cell-based exontrap assay. Results We identified 19 different SERPINC1 abnormalities, including 8 novel mutations, in 21 Japanese patients with AT deficiency. These abnormalities were distributed as follows: 9 missense mutations (42.9%), 3 nonsense mutations (14.3%), 1 splice-site mutation (4.8%), 2 small insertions (9.5%), 2 deletion mutations (9.5%) and 4 large deletions (19.0%). Cases with large deletions of SERPINC1 included Alu-mediated gene rearrangements and non-Alu-mediated complex gene rearrangements; the latter could conceivably be explained using the fork stalling and template switching (FoSTeS) model. Conclusions We identified a variety of SERPINC1 defects in Japanese patients with AT deficiency. The SERPINC1 mutations detected in patients with type I AT deficiency included single nucleotide missense or nonsense mutations, small intragenic insertions or deletions, and large genomic structural deletions. Large deletions of SERPINC1 were caused by various recurrent or non-recurrent complex genomic rearrangement mutations.

Some genetic and treatment-related factors are risk factors for inhibitor development in patients with hemophilia A (PwHA). However, the genotype distribution of the factor VIII gene (F8) and genetic impact on inhibitor development in Japanese PwHA remain unknown. In 2007, the Japan Hemophilia Inhibitor Study 2 (J-HIS2) was organized to establish a nationwide registry system for hemophiliacs and to elucidate risk factors for inhibitor development, designed for prospective investigation following a retrospective study (J-HIS1) which had already finished. Patients, newly diagnosed after January 2007, were enrolled in J-HIS2 and followed up for inhibitor development and clinical environments since 2008 onward. In the present study, F8 genotypes of PwHA were investigated in the patients recruited from the J-HIS2 cohort as well as those with inhibitor from the J-HIS1 cohort. F8 variants identified in 59 PwHA with inhibitor in J-HIS1 were: 20 intron-22 inversions, 5 intron-1 inversions, 9 large deletions, 4 nonsense, 8 missense, 11 small in/del, and 2 splice-site variants. F8 variants identified in 267 (67 with inhibitor) PwHA in J-HIS2 were: 76(28) intron–22 inversions, 3(2) intron–1 inversion, 1(0) duplication, 8(5) large deletions, 21(7) nonsense, 109(7) missense, 40(11) small in/del, and 9(7) splice-site variants. Forty variants were novel. The cumulative inhibitor incidence rate in the severe group with null changes was 42.4% (95% confidence interval [CI]: 33.7–50.8), higher than that with nonnull changes (15.6% [95%CI: 6.8–27.8]), in J-HIS2. Relative risk for inhibitor development of null changes was 2.89. The spectrum of F8 genotype and genetic impact on inhibitor development in Japanese PwHA were consistent with the previous reports.

Bone marrow development and endochondral bone formation occur simultaneously. During endochondral ossification, periosteal vasculatures and stromal progenitors invade the primary avascular cartilaginous anlage, which induces primitive marrow development. We previously determined that bone marrow podoplanin (PDPN)-expressing stromal cells exist in the perivascular microenvironment and promote megakaryopoiesis and erythropoiesis. In this study, we aimed to examine the involvement of PDPN-expressing stromal cells in postnatal bone marrow generation. Using histological analysis, we observed that periosteum-derived PDPN-expressing stromal cells infiltrated the cartilaginous anlage of the postnatal epiphysis and populated on the primitive vasculature of secondary ossification center. Furthermore, immunophenotyping and cellular characteristic analyses indicated that the PDPN-expressing stromal cells constituted a subpopulation of the skeletal stem cell lineage. In vitro xenovascular model cocultured with human umbilical vein endothelial cells and PDPN-expressing skeletal stem cell progenies showed that PDPN-expressing stromal cells maintained vascular integrity via the release of angiogenic factors and vascular basement membrane-related extracellular matrices. We show that in this process, Notch signal activation committed the PDPN-expressing stromal cells into a dominant state with basement membrane-related extracellular matrices, especially type IV collagens. Our findings suggest that the PDPN-expressing stromal cells regulate the integrity of the primitive vasculatures in the epiphyseal nascent marrow. To the best of our knowledge, this is the first study to comprehensively examine how PDPN-expressing stromal cells contribute to marrow development and homeostasis.

Background Hemophilia A (HA) is an X‐linked recessive bleeding disorder caused by pathogenic variants of the coagulation factor VIII gene (F8). Half of the patients with severe HA have a recurrent inversion in the X chromosome, that is, F8 intron 22 or intron 1 inversion. Here, we characterized an abnormal F8 due to atypical complex X chromosome rearrangements in a Japanese patient with severe HA. Methods Recurrent F8 inversions were tested with inverse shifting‐PCR. The genomic structure was investigated using PCR‐based direct sequencing or quantitative PCR. Results The proband's X chromosome had a 119.5 kb insertion, a reverse duplex of an extragenic sequence on the F8 telomere region into the F8 intron 1 with two breakpoints. The telomeric breakpoint was a joining from the F8 intron 1 to the inverted FUNDC2 via a two‐base microhomology, and the centromeric breakpoint was a recombination between F8 intron 1 homologous sequences. The rearrangement mechanism was suggested as a multi‐step rearrangement with template switching such as fork stalling and template switching (FoSTeS)/microhomology‐mediated break‐induced replication (MMBIR) and/or homologous sequence‐associated recombination during a sister chromatid formation. Conclusion We identified the aberrant X chromosome with a split F8 due to a multi‐step rearrangement in a patient with severe HA., Hemophilia A (HA) is an X‐linked recessive bleeding disorder caused by mutations in the coagulation factor VIII gene (F8). We identified the aberrant chromosome X with a split F8 due to a multi‐step rearrangement in a severe HA patient. The rearrangement mechanism was suggested as a multi‐step rearrangement with template switching such as FoSTeS/MMBIR and/or homologous sequence associating recombination during a sister chromatid formation.

Introduction Antithrombin resistance (ATR) prothrombinemia is an inherited thrombophilic disorder caused by missense mutations in prothrombin gene (F2) at Arg596 of the sodium-binding region. Previously, prothrombin mutants Yukuhashi (Arg596Leu), Belgrade (Arg596Gln), and Padua 2 (Arg596Trp) were reported as ATR-prothrombins possessing a risk of familial venous thrombosis. To identify additional F2 mutations causing the ATR-phenotype, we investigated the coagulant properties of recombinant prothrombins mutated at amino acid residues within the sodium-binding region by single nucleotide substitutions (Thr540, Arg541, Glu592, and Lys599). Materials and methods We constructed expression vectors of prothrombin mutants, established stably transfected HEK293 cells, and isolated the recombinant prothrombin proteins. We evaluated procoagulant activity and ATR-phenotypes of those mutants in reconstituted plasma by mixing with prothrombin deficient plasma. Results The secreted quantity of all prothrombin mutants was the same as that of the wild-type prothrombin. Procoagulant activity of each mutant varied from 1.7% to 79.5% in a one-stage clotting assay and from 2.0% to 104.5% in a two-stage chromogenic assay. Most prothrombin mutants tested presented with a severe ATR-phenotype. To estimate the thrombosis risk of these mutations, we determined the residual clotting activity (RCA) after 30 min inactivation with antithrombin. RCA scores, normalized to the wild-type, revealed that prothrombin mutants Lys599Arg (5.35) and Glu592Gln (4.71) had high scores, which were comparable with prothrombins Yukuhashi (4.36) and Belgrade (5.19). Conclusions Mutation of prothrombin at the sodium-binding site caused ATR-phenotypes. Of those tested, Lys599Arg and Glu592Gln may possess a thrombosis risk as large as the known pathogenic prothrombins Yukuhashi and Belgrade.

Idiopathic portal hypertension (IPH), causing aberrant portal hemodynamics, is a disease with an as yet unidentified cause and no treatment has been established so far. The Japanese research group on IPH in Japan was set up in 1975 by the Ministry of Health, Labour and Welfare, furthermore extra-hepatic portal obstruction (EHO) and Budd-Chiari syndrome (BCS) have been added to the research subjects and further work has continued. The aims of the research group are to accurately evaluate the current status of the three diseases in Japan, elucidate the disease aetiology and pathogenesis, and develop new treatments. Due to the long-term efforts of the Japanese research group, aberrant portal hemodynamics has been investigated to date in a variety of aspects, from epidemiological and pathological studies to molecular biology analysis. As a result, it has been shown that there are abnormal genes in the liver, specific for IPH. In addition, pathological findings of BCS were internationally compared and the difference in findings between Japan and Western countries has been clarified. Furthermore, it was found that complication rates of hepatocellular carcinoma (HCC) in BCS were higher in Japan. Based on the research, "Diagnosis and treatment of aberrant portal hemodynamics (2001)", including diagnostic criteria for aberrant portal hemodynamics, was published in 2001. In 2013, it was revised to "Diagnosis and treatment guidelines for aberrant portal hemodynamics (2013)" after the incorporation of diagnosis and treatment in accordance with its current status. This article is protected by copyright. All rights reserved.

Syndecan-4 is a transmembrane heparan sulfate proteoglycan expressed in a variety of cells, and its heparan sulfate glycosaminoglycan side chains bind to several proteins exhibiting various biological roles. The authors have previously demonstrated syndecan-4&prime, s critical roles in pulmonary inflammation. In the current study, however, its role in pulmonary fibrosis was evaluated. Wild-type and syndecan-4-deficient mice were injected with bleomycin, and several parameters of inflammation and fibrosis were analyzed. The mRNA expression of collagen and &alpha, smooth muscle action (&alpha, SMA) in lung tissues, as well as the histopathological lung fibrosis score and collagen content in lung tissues, were significantly higher in the syndecan-4-deficient mice. However, the total cell count and cell differentiation in bronchoalveolar lavage fluid were equivalent between the wild-type and syndecan-4-deficient mice. Although there was no difference in the TGF-&beta, expression in lung tissues between the wild-type and syndecan-4-deficient mice, significantly more activation of Smad3 in lung tissues was observed in the syndecan-4-deficient mice compared to the wild-type mice. Furthermore, in the in vitro experiments using lung fibroblasts, the co-incubation of syndecan-4 significantly inhibited TGF-&beta, induced Smad3 activation, collagen and &alpha, SMA upregulation. Moreover, syndecan-4 knock-down by siRNA increased TGF-&beta, induced Smad3 activation and upregulated collagen and &alpha, SMA expression. These findings showed that syndecan-4 inhibits the development of pulmonary fibrosis, at least in part, through attenuating TGF-&beta, signaling.

Bone marrow (BM), the tissue specializing in the production of hematopoietic cells, consists of multiple components (e.g., extracellular matrixes, vasculatures, and stromal cells) that generate a complex three-dimensional network and several localized microenvironment. These microenvironments regulate hematopoietic stem and progenitor cells, including megakaryocyte lineage cells. In this review, we first provide an overview of the microenvironment for hematopoietic stem cells as an introduction to bone marrow microenvironment and subsequently summarize the microenvironment for megakaryocyte differentiation and maturation (megakaryopoiesis). In the last portion, we describe megakaryocyte regulation by podoplanin-positive peri-arteriolar stromal cells in the mouse bone marrow.

Introduction Hemophilia B is an X-linked recessive bleeding disorder caused by coagulation factor IX (FIX) gene (F9) mutations. Several F9 synonymous mutations have been known to cause hemophilia B; however, the deleterious mechanisms underlying the development of hemophilia B have not been completely understood. To elucidate the molecular pathogenesis causing hemophilia B, we investigated the synonymous F9 mutation: c.87A>G, p.(Thr29=). Materials and methods The influence of F9 c.87A>G on mRNA splicing was analyzed by exon-trap assay and in silico prediction. We prepared FIX expression vectors using mutant F9 cDNA and transfected HepG2 cells to investigate intracellular transport and extracellular secretion of FIX. Intracellular kinetics of the expressed FIX was examined by treatment with the proteasome inhibitor MG132. Results Exon-trap analysis revealed that F9 c.87A>G resulted in almost (99.1%) aberrant splicing (r.83_88del). In silico analysis predicted that F9 c.87A>G influenced the splicing pattern by generating an available aberrant 5′ splice site. The aberrant F9 mRNA (r.83_88del) was translated to a mutant FIX p.Cys28_Val30delinsPhe with an in-frame mutation at the signal peptide cleavage site. Simultaneously, a small amount (0.9%) of mutant F9 r.87A>G translating into WT FIX p.Thr29 = was also observed. The mutant FIX was abnormally retained in the endoplasmic reticulum (ER) and was not extracellularly secreted. It appeared to be intracellularly degraded via proteasome-dependent degradation machinery. Conclusion F9 c.87A>G was found to cause abnormal mRNA splicing, r.83_88del, and produce the mutant FIX p.Cys28_Val30delinsPhe. The mutant FIX is an abnormal protein with extracellular secretory defects and is intracellularly eliminated via proteasome-dependent ER-associated degradation.

Introduction Use of combined oral contraceptives (COCs) results in acquired protein S (PS) deficiency, a well-established risk factor for venous thromboembolism (VTE). The risk of VTE due to COCs containing newer-generation progestins is double compared with COCs containing older-generation progestins, although there is little difference in estrogen contents between the generations. In contrast, progestin-only contraceptives do not confer an increased risk of VTE. In this study, we aimed to investigate how different isoforms of progestin in COCs affect the risk of VTE by measuring PS expression. Materials and methods The effect of progestin, levonorgestrel (LNG) or drospirenone (DRSP), on PS mRNA expression in HepG2 cells was measured using reverse transcription-quantitative PCR; PS level was determined using Western blot analysis. PROS1 promoter activity, PS mRNA stability, and de novo synthesis of PS mRNA were examined in HepG2 cells after treatment with progestin. Results and conclusions In the presence of progestins, PS mRNA and protein expressions were significantly upregulated in HepG2 cells due to the augmentation of de novo PS mRNA expression modulated by RNA polymerase II (Pol II), thereby facilitating PS transcription elongation. Moreover, the transcription elongation inhibitor blocked progestin-mediated de novo PS mRNA expression. Conversely, progestin did not affect PROS1 promoter activity and PS mRNA stability. Pol II elongation efficiency in the newer-generation progestin (DRSP) treatment was not as strong compared with older-generation progestin (LNG), suggesting the difference in VTE risk between COC generations.

The use of lung progenitors for regenerative medicine appears promising, but their biology is not fully understood. Here, we found anti-inflammatory attributes in bronchiolar progenitors that were sorted as a multipotent subset of mouse club cells and found to express secretory leukocyte protease inhibitor (SLPI). Notably, the impaired expression of SLPI in mice increased the number of bronchiolar progenitors and decreased the lung inflammation. We determined a transcriptional profile for the bronchiolar progenitors of Slpi-deficient mice and identified syndecan 4, whose expression was markedly elevated as compared to that of wild-type mice. Systemic administration of recombinant syndecan 4 protein caused a substantial increase in the number of bronchiolar progenitors with concomitant attenuation of both airway and alveolar inflammation. The syndecan 4 administration also resulted in activation of the Keap1-Nrf2 antioxidant pathway in lung cells, which is critically involved in the therapeutic responses to the syndecan 4 treatment. Moreover, in 3D culture, the presence of syndecan 4 induced differentiated club cells to undergo Nrf2-dependent transition into bronchiolar progenitors. Our observations reveal that differentiative switches between bronchiolar progenitors and club cells are under the Nrf2-mediated control of SLPI and syndecan 4, suggesting the possibility of new therapeutic approaches in inflammatory lung diseases.

We describe the case of a 45-year-old man with congenital thrombophilia induced by antithrombin resistance. He had recurrent venous thrombosis without traditional risk factors or abnormal coagulation function and had a family history of venous thrombosis which included his mother, brother, and nephew. We suspected the association of hereditary antithrombin resistance, which has been reported in some cases of familial venous thromboses due to prothrombin mutations. Although prothrombin abnormality typically shows a bleeding tendency, variations of arginine at position 596 in the gene encoding prothrombin have been reported to conversely cause thrombosis. Therefore, we tested and detected antithrombin resistance in the patient's plasma. We also performed genetic analysis for his second filial generation, and found a missense mutation (c.1787G>A), resulting in a substitution of arginine for glutamine at position 596 (p.Arg596Gln) in the gene encoding prothrombin (called prothrombin Belgrade). The Gln596 substitution caused the susceptibility to thrombosis. This variation is the same as one previously reported in Serbia and India, and it is the third report in Japan. .

Von Willebrand factor (VWF) is synthesized in megakaryocytes and endothelial cells (ECs) and has two main roles: to carry and protect coagulation factor VIII (FVIII) from degradation by forming VWF–FVIII complex; and to mediate platelet adhesion and aggregation at sites of vascular injury. Previous research using the HEK293 cell line revealed that the VWF K1362 mutation interacted directly with platelet glycoprotein Ib (GPIb). Vwf K1362A knock-in (KI) mice were therefore generated to verify the in vivo function of residue 1362 in binding to platelet GPIb. The Cre-loxP system was employed to introduce the Vwf K1362A mutation systemically in mice. In blood coagulation analysis, the VWF antigen (VWF:Ag) of Lys1362Ala KI homozygous (homo) mice was below the sensitivity of detection by enzyme-linked immunosorbent assay. FVIII activities (FVIII:C) were 47.9 ± 0.3 and 3.3 ± 0.3% (K1362A heterozygous (hetero) and K1362A KI homo mice, respectively) compared to wild-type mice. Immunohistochemical staining analysis revealed that VWF protein did not exist in ECs of K1362A KI homo mice. These results indicated that VWF protein synthesis of K1362A was impaired after transcription in mice. K1362 seems to represent a very important position not only for VWF function, but also for VWF synthesis in mice.

Sphingosine kinases (SPHK) are important to determine cells' fate by producing sphingosine 1-phosphate. Reportedly, exogenous SPHK2 overexpression induces cell cycle arrest or cell death. However, the regulatory mechanism of SPHK2 expression has not been fully elucidated. Here, we analyzed this issue using human colon cancer cell lines under various stress conditions. Serum depletion (FCS(-)) but not hypoxia and glucose depletion increased mRNA, protein and enzyme activity of SPHK2 but not SPHK1. In HCT116 cells mostly used, SPHK2 activity was predominant over SPHK1, and serum depletion increased both nuclear and cytoplasmic SPHK2 activity. Based on previous reports analyzing cellular response after serum depletion, the temporal changes of intracellular signaling molecules and candidate transcription factors for SPHK2 were examined using serum-depleted HCT116 cells, and performed transfection experiments with siRNA or cDNA of candidate transcription factors. Results showed that the rapid and transient JNK activation followed by CREB activation was the major regulator of increased SPHK2 transcription in FCS(-) culture. EMSA and ChIP assay confirmed the direct binding of activated CREB to the CREB binding site of 5' SPHK2 promoter region. Colon cancer cells examined continued to grow in FCS(-) culture, although mildly, while hypoxia and glucose depletion suppressed cell proliferation or induced cell death, suggesting the different role of SPHK2 in different stress conditions. Because of the unique relationship observed after serum depletion, we examined effects of siRNA for SPHK2, and found the role of SPHK2 as a growth or survival factor but not a cell proliferation inhibitor in FCS(-) culture.

BACKGROUND Syndecan-4 is a transmembrane heparan sulfate proteoglycan expressed in a variety of cells, and glycosaminoglycan side chains of syndecan-4 bind to several proteins, suggesting several biological functions. However, the role of syndecan-4 in acute bacterial pneumonia has not yet been elucidated. METHODS Serum syndecan-4 levels were measured in patients with acute pneumonia, and the relationships between serum syndecan-4 levels and clinical parameters were analyzed. Next, we treated wild-type and syndecan-4-deficient mice with Streptococcus pneumoniae intranasally and analyzed the phenotype of syndecan-4-deficient mice. RESULTS In the patients with acute pneumonia, serum syndecan-4 levels were significantly higher than in the healthy volunteers and correlated negatively with the pneumonia severity score. In addition, in patients who improved with short-term antibiotic therapy, serum syndecan-4 levels were higher on admission and gradually increased during antibiotic therapy. Furthermore, in syndecan-4-deficient mice, the survival rate was significantly worse, and total neutrophil counts in bronchoalveolar lavage fluid, bacterial counts in blood, and plasma levels of inflammatory cytokines were significantly higher than in wild-type mice. CONCLUSIONS These results suggest that syndecan-4 has an anti-inflammatory function in acute pneumonia and could serve as a useful biomarker in these patients.

Acid ceramidase (ACDase) metabolizes ceramide to sphingosine, leading to sphingosine 1-phosphate production. Reportedly, ACDase has been upregulated in prostate cancer. However, its regulatory mechanism remains unclear. LNCaP (androgen-sensitive prostate cancer cell line) but not PC3 and DU-145, (androgen-unresponsive cell lines) exhibited the highest ACDase protein. Among three cell lines, ASAH1 mRNA level was not correlated with ACDase protein expression, and the 5'-promoter activity did not show androgen dependency, suggesting the post-transcriptional regulation of ACDase in LNCaP cells. Based on these results, LNCaP was analysed further. Casodex, androgen receptor antagonist, and charcoal-stripped FCS (CS-FCS) decreased ACDase protein and activity, whereas dihydrotestosterone in CS-FCS culture increased ACDase protein and enzyme activity. MG132, a proteasome inhibitor, prevented the decrease of ACDase protein when cultured in CS-FCS, suggesting the involvement of ubiquitin/proteasome system. Reportedly, USP2, a deubiquitinase, plays an important role in LNCaP cells. USP2 siRNA decreased ACDase protein, whereas USP2 overexpression increased ACDase protein of LNCaP cells. However, SKP2, an ubiquitin E3 ligase known to be active in prostate cancer, did not affect androgen-dependent ACDase expression in LNCaP cells. Thus, ACDase regulation by androgen in androgen-sensitive LNCaP cells is mainly due to its prolonged protein half-life by androgen-stimulated USP2 expression.

Introduction Antithrombin resistance (ATR) is a novel thrombotic risk in abnormal prothrombins. A manual ATR assay using Oxyuranus scutellatus (Ox) venom as a prothrombin activator was established for detecting antithrombin-resistant prothrombin. However, this assay was limited because of Ox snake venom availability and its throughput capacity. Here, we have improved the ATR assay using bovine factors Xa and Va (FXa/Va) as prothrombin activators and have optimised assay conditions for an automated instrument (ACL TOP 500). Methods Diluted plasma was incubated with a prothrombin activator mix (phospholipids, CaCl2 , and bovine FXa/Va), followed by inactivation with antithrombin for 10, 20 and 30 minutes. We added a chromogenic substrate S-2238, and assessed changes in absorbance/min at 405 nm. We also adapted assay conditions for ACL TOP 500. Results Optimum conditions for FXa/Va treatment were 6.25% phospholipids, 5 mM CaCL2 , 0.01 μg/mL FXa and 0.1 μg/mL FVa. ATR assay kinetics with the FXa/Va activator was comparable with that with the Ox activator in heterozygous reconstituted plasma with the recombinant wild-type or antithrombin-resistant prothrombin. Using ACL TOP 500, optimum conditions for the FXa/Va treatment were 10.0% phospholipids, 5 mM CaCl2 , 0.02 μg/mL FXa and 0.2 μg/mL FVa. The automated ATR assay with the FXa/Va activator demonstrated good detectability for antithrombin-resistant prothrombin in plasma from a heterozygous carrier with prothrombin Yukuhashi or Belgrade. Conclusion We optimised the ATR assay with the FXa/Va activator and adapted the assay for ACL TOP 500; the assay showed the ability to clearly detect antithrombin-resistant prothrombin in manual and automated procedures.

Introduction Prothrombin Yukuhashi (p.Arg596Leu) mutation can result in thrombophilia associated with antithrombin (AT) resistance. Mutant thrombin, an active form of prothrombin Yukuhashi, demonstrated moderately lower clotting activity than the wild-type but substantially impaired the formation of the complex with AT. However, the effects of the mutation on the thrombomodulin (TM)–protein C (PC) anticoagulant system have not been previously elucidated. Materials and Methods We prepared recombinant wild-type and mutant prothrombins, converted to thrombins using Oxyuranus scutellatus venom, and performed fibrinogen-clotting assays with or without recombinant soluble TM (rTM). We also evaluated activated PC (APC) generation activity of recombinant thrombins by measuring APC activity after incubation with human PC in the presence or absence of rTM. Result and Conclusions rTM treatment reduced the relative fibrinogen-clotting activity of the wild-type down to 8.4% in a concentration-dependent manner, whereas the activity of the mutant was only decreased to 44%. In the absence of rTM, APC generation activity (∆A/min at 405 nm) was fairly low (0.0089 for the wild-type and 0.0039 for the mutant). In the presence of rTM, however, APC generation activity was enhanced to 0.0907 (10.2-fold) for the wild-type and to 0.0492 (12.6-fold) for the mutant, and the relative activity of the mutant with rTM was 54% of that of the wild-type. These data suggested that the prothrombin Yukuhashi mutation may cause TM resistance in terms of inhibition of fibrinogen clotting; this may contribute to susceptibility to thrombosis, although the enhancing effect of APC generation can be maintained.

Multidrug resistance (MDR) is a serious problem faced in the treatment of malignant tumors. In this study, we characterized the expression of non-homologous DNA end joining (NHEJ) components, a major DNA double strand break (DSB) repair mechanism in mammals, in K562 cell and its daunorubicin (DNR)-resistant subclone (K562/DNR). K562/DNR overexpressed major enzymes of NHEJ, DNA-PKcs and DNA ligase IV, and K562/DNR repaired DSB more rapidly than K562 after DNA damage by neocarzinostatin (MDR1-independent radiation-mimetic). Overexpressed DNA-PKcs and DNA ligase IV were also observed in DNR-resistant HL60 (HL60/DNR) cells as compared with parental HL60 cells. Expression level of DNA-PKcs mRNA paralleled its protein level, and the promoter activity of DNA-PKcs of K562/DNR was higher than that of K562, and the 5'-region between -49bp and the first exon was important for its activity. Because this region is GC-rich, we tried to suppress Sp1 family transcription factor using mithramycin A (MMA), a specific Sp1 family inhibitor, and siRNAs for Sp1 and Sp3. Both MMA and siRNAs suppressed DNA-PKcs expression. Higher serine-phosphorylated Sp1 but not total Sp1 of both K562/DNR and HL60/DNR was observed compared with their parental K562 and HL60 cells. DNA ligase IV expression of K562/DNR was also suppressed significantly with Sp1 family protein inhibition. EMSA and ChIP assay confirmed higher binding of Sp1 and Sp3 with DNA-PKcs 5'-promoter region of DNA-PKcs of K562/DNR than that of K562. Thus, the Sp1 family transcription factor affects important NHEJ component expressions in anti-cancer drug-resistant malignant cells, leading to the more aggressive MDR phenotype.

Sphingosine kinase 1 (SPHK1) overexpression in malignant cells has been reported. Mouse Friend cells showed higher SPHK1 but not SPHK2 expression compared with other mouse cell lines. A Sphk1 promoter analysis demonstrated the region between − 53 bp and the first exon as the minimal promoter. Further promoter truncation revealed the importance of a MYB-binding site. EMSA using this region as the probe demonstrated one band containing c-MYB protein, and its intensity decreased during erythroid differentiation with hexamethylane bisacetamide (HMBA), a potent inducer of erythroid differentiation of Friend cells. ChIP assay also revealed in vivo binding of c-MYB. c-MYB overexpression and siRNA for c-Myb affected SPHK1 expression, confirming the important regulatory role of c-MYB in SPHK1 expression. HMBA reduced c-MYB expression rapidly. Induced differentiation by HMBA caused a marked and rapid reduction of SPHK1 mRNA, protein and enzyme activity leading to the rapid decrease of cellular sphingosine 1-phosphate level. Moreover, terminally differentiated cells did not resume SPHK1 expression. Compared with original Friend cells, stable overexpression of wild-type SPHK1 showed higher cell proliferation, resistance to cell death by serum depletion. Interestingly, HMBA-induced differentiation of these cells was delayed but not completely suppressed. In contrast, SPHK inhibitor and its siRNA inhibited cell growth and enhanced HMBA-induced differentiation significantly, suggesting that SPHK1 delayed HMBA-induced differentiation by its cell proliferation-promoting activity. Effects of pertussis toxin, a G-protein-coupled receptor inhibitor, and S1P receptor antagonist on Friend cell growth and differentiation were negligible, suggesting the importance of the intracellular SPHK1/S1P signaling in Friend cells.

Syndecan-4, a cell-surface heparan sulfate proteoglycan, can participate in inflammation and wound healing as a host defense molecule. Tumour necrosis factor (TNF)-α, one of the most potent proinflammatory cytokines, is known to upregulate syndecan-4 expression, but the precise mechanisms are unclear. To elucidate these mechanisms in detail, we examined syndecan-4 upregulation by TNF-α in the endothelium-like EAhy926 cell. Of the two putative nuclear factor kappa-B (NF-κB) binding sites in the syndecan-4 gene (SDC4) promoter, deletion or mutation of one or both sites significantly diminished the effects of TNF-α. Electrophoretic mobility shift assays showed that p65 and c-Rel, but not p50, bound to these NF-κB binding sites, whereas pull-down assays showed binding of all three NF-κB components. Chromatin immunoprecipitation assays clearly showed that p65 and phosphorylated p65, but not p50 or c-Rel, bound to the SDC4 promoter. An NF-κB inhibitor, p65 knockdown and a transcriptional elongation inhibitor completely blocked the effect of TNF-α on SDC4 promoter activity and significantly, but not completely, blocked that on SDC4 mRNA expression. These data suggest that NF-κB p65 could be a key mediator of syndecan-4 upregulation by TNF-α through two binding sites in the SDC4 promoter, but other NF-κB-p65 independent pathways might also be involved through transcriptional elongation.

Essentials Prothrombin Belgrade mutation leads to antithrombin resistance. Clinical and biochemical phenotypes in a large family with this mutation were investigated. In carriers, we detected decreased factor II activity and increased endogenous thrombin potential. Prothrombin Belgrade mutation represents a strong prothrombotic risk factor. SummaryBackground The recently reported c.1787G>A mutation in the prothrombin gene leads to Arg596Gln replacement in the protein molecule (prothrombin Belgrade). This substitution impairs binding of antithrombin to thrombin and results in inherited thrombophilia, known as antithrombin resistance. Objectives We aimed to elucidate the clinical and biochemical characteristics of thrombophilia associated with antithrombin resistance in a large Serbian family with the prothrombin Belgrade mutation. Patients and methods Nineteen family members were investigated, among whom 10 were carriers of the c.1787G>A mutation. In all subjects the clinical phenotype was determined and laboratory investigations of hemostatic parameters were performed. Results Six out of the 10 mutation carriers developed thromboembolic events, mainly deep venous and mesenteric vein thrombosis. The median age of the first thrombotic event was 26.5 (12–41) years, whereas the incidence rate of first thrombosis was 2.2% per year. In all mutation carriers prothrombin activity was significantly decreased in comparison with non-carriers, clearly distinguishing each group. However, the presence of the mutation did not affect the prothrombin antigen level in plasma. The endogenous thrombin potential was significantly increased in all carriers in comparison with non-carriers, indicating the presence of blood hypercoagulability. Interestingly, levels of D-dimer and the F1+2 fragment were similar in both groups. Conclusions Although rare, the prothrombin Belgrade mutation represents strong thrombophilia with early onset of thrombosis in the investigated family. According to our results, decreased prothrombin activity may be a simple screening test for detection of this mutation in thrombotic patients.

Hemophilia B Leyden is a unique subtype of hemophilia B, characterized by increasing factor IX activity (FIX:C) after puberty and a lower normal range of FIX:C throughout adulthood. However, to date, no Japanese case has been reported. Here, we report a case of hemophilia B Leyden in a 22-year-old male. He suffered from subgaleal hematoma, and was subsequently diagnosed with hemophilia B (FIX:C 0.2%) in the neonatal period. Both his parents are Japanese. There was no history of hemophilia in his family. FIX:C gradually increased with age (8% at age = 1; 14% at age = 7; 19% at age = 12; 32% at age = 18). FIX:C is within the range 30-40% in recent several years. He once required administration of FIX concentrate against traumatic tongue bleeding at 7 years of age. Genotyping analysis of FIX was performed after informed consent at 21 years of age, and a point mutation (c.-35G>A) was detected. This mutation has been reported previously as the Leyden mutation. Although it has been reported that hemophilia B Leyden is seen in 1.9% of patients with hemophilia B, the present case is the first report of hemophilia B Leyden from Japan.